The present invention relates to methods for attaching target molecules such as oligonucleotides (oligos) to a surface, and to compositions for use in such methods. In another aspect, the invention relates to the resultant coated surfaces themselves. In yet another aspect, the invention relates to the use of photochemical and thermochemical means to attach molecules to a surface.
The immobilization of deoxyribonucleic acid (DNA) onto support surfaces has become an important aspect in the development of DNA-based assay systems as well as for other purposes, including the development of microfabricated arrays for DNA analysis. See, for instance, xe2x80x9cMicrochip Arrays Put DNA on the Spotxe2x80x9d, R. Service, Science 282(5388):396-399, 16 October 1998; and xe2x80x9cFomenting a Revolution, in Miniaturexe2x80x9d, I. Amato, Science 282(5388): 402-405, Oct. 16, 1998.
See also, xe2x80x9cThe Development of Microfabricated Arrays of DNA Sequencing and Analysisxe2x80x9d, O""Donnell-Maloney et al., TIBTECH 14:401-407 (1996). Generally, such procedures are carried out on the surface of microwell plates, tubes, beads, microscope slides, silicon wafers or membranes. Certain approaches, in particular, have been developed to enable or improve the likelihood of end-point attachment of a synthetic oligonucleotide to a surface. End-point attachment (i.e., with the nucleic acid sequence attached through one or the other terminal nucleotide) is desirable because the entire length of the sequence will be available for hybridization to another nucleic acid sequence. This is particularly advantageous for the detection of single base pair changes under stringent hybridization conditions.
Hybridization is the method used most routinely to measure nucleic acids by base pairing to probes immobilized on a solid support. When combined with amplification techniques such as the polymerase chain reaction (PCR) or ligase chain reaction (LCR), hybridization assays are a powerful tool for diagnosis and research. Microwell plates, in particular, are convenient and useful for assaying relatively large numbers of samples. Several methods have been used for immobilization of nucleic acid probes onto microwell plates. Some of these involve adsorption of unmodified or modified oligonucleotides onto polystyrene plates. Others involve covalent immobilization. Various methods have also been used to increase the sensitivity of hybridization assays. Polymeric capture probes (also known as target molecules) and detection probes have been synthesized and used to obtain sensitivities down to 107 DNA molecules/ml. Another method used branched oligonucleotides to increase the sensitivity of hybridization assays. Yet another method used a multi-step antibody-enhanced method. Other types of nucleic acid probes such as ribonucleic acid (RNA), complementary DNA (cDNA) and peptide nucleic acids (PNA""s) have also been immobilized onto microwell plates for hybridization of PCR products in diagnostic applications. Furthermore, PCR primers have been immobilized onto microwell plates for solid phase PCR.
Only a relative few approaches to immobilizing DNA, to date, have found their way into commercial products. One such product is known as xe2x80x9cNucleoLink(trademark)xe2x80x9d, and is available from Nalge Nunc International (see, e.g., Nunc Tech Note Vol. 3, No. 17). In this product, the DNA is reacted with a carbodiimide to activate 5xe2x80x2-phosphate groups which then react with functional groups on the surface. Disadvantages of this approach are that it requires the extra step of adding the carbodiimide reagent as well as a five hour reaction time for immobilization of DNA, and it is limited to a single type of substrate material.
As another example, Pierce has recently introduced a proprietary DNA immobilization product known as xe2x80x9cReacti-Bind(trademark) DNA Coating Solutionsxe2x80x9d (see xe2x80x9cInstructionsxe2x80x94Reacti-Bind(trademark) DNA Coating Solutionxe2x80x9d 1/1997). This product is a solution that is mixed with DNA and applied to surfaces such as polystyrene or polypropylene. After overnight incubation, the solution is removed, the surface washed with buffer and dried, after which it is ready for hybridization. Although the product literature describes it as being useful for all common plastic surfaces used in the laboratory, it does have some limitations. For example, Applicants were not able to demonstrate useful immobilization of DNA onto polypropylene using the manufacturer""s instructions. Furthermore, this product requires large amounts of DNA. The instructions indicate that the DNA should be used at a concentration between 0.5 and 5 xcexcg/ml.
Similarly, Costar sells a product called xe2x80x9cDNA-BINDTMxe2x80x9d for use in attaching DNA to the surface of a well in a microwell plate (see, e.g., the DNA-BINDTM xe2x80x9cApplication Guidexe2x80x9d). The surface of the DNA-BIND(trademark) plate is coated with an uncharged, nonpolymeric heterobifunctional reagent containing an N-oxysuccinimide (NOS) reactive group. This group reacts with nucleophiles such as primary amines. The heterobifunctional coating reagent also contains a photochemical group and spacer arm which covalently links the reactive group to the surface of the polystyrene plate. Thereafter, amine-modified DNA can be covalently coupled to the NOS surface. The DNA is modified by adding a primary amine either during the synthesis process to the nascent oligomer or enzymatically to the preformed sequence. Since the DNA-BIND(trademark) product is polystyrene based, it is of limited use for those applications that require elevated temperatures such as thermal cycling.
These various products may be useful for some purposes, or under certain circumstances, but all tend to suffer from one or more drawbacks and constraints. In particular, they either tend to require large amounts of oligonucleotide, render background noise levels that are unsuitably high and/or lack versatility.
International Patent Application No. PCT/US98/20140, assigned to the assignee of the present application, describes and claims, inter alia, a reagent composition for attaching a target molecule to the surface of a substrate, the reagent composition comprising one or more groups for attracting the target molecule to the reagent, and one or more thermochemically reactive groups for forming covalent bonds with corresponding functional groups on the attracted target molecule. Optionally, the composition further provides photogroups for use in attaching the composition to a surface. In one embodiment, for instance, a plurality of photogroups and a plurality of cationic groups (in the form of quaternary ammonium groups) are attached to a hydrophilic polymer backbone. This polymer can then be coimmobilized with a second polymer backbone that provides the above-described thermochemically reactive groups (e.g., N-oxysuccinimide (xe2x80x9cNOSxe2x80x9d) groups) for immobilization of target molecules.
While reagent compositions having both attracting groups and thermochemically reactive groups, as described in the above-captioned PCT application, remain useful and preferred for many applications, Applicants also find that the attracting groups may not be required under all circumstances. For instance, one suitable process for preparing activated slides for microarrays includes the steps of coating the slides with a reagent composition of a type described in the PCT application (and particularly, one having both attracting groups as well as photoreactive and thermochemically reactive groups). The polymers are attached to the slide by activation of the photoreactive groups, following by the application of small volumes (e.g., several nanoliters or less) of target molecules (e.g., oligonucleotides) using precision printing techniques.
Once applied, the solvent used to deliver the oligonucleotide is dried (as the oligonucleotides are attracted to the bound polymer), and the slide incubated under conditions suitable to permit the thermochemical coupling of the oligonucleotide to the bound polymer. Thereafter, however, any unbound oligonucleotide is typically washed off of the slide. Applicants have found, however, that there occasionally remains a detectable trail of unbound oligonucleotide, referred to as a xe2x80x9ccomet effectxe2x80x9d, leading away from the spot. This trail is presumably due to the attractive forces within the bound polymer present on the slide surface that surrounds the spot, serving to tie up the generally negatively charged oligonucleotide as it is washed from the spot. This trail, in turn, can provide undesirable and unduly high levels of background noise.
Applicants have found that under such circumstances (e.g., the application of small volumes directly to a generally flat surface) polymeric reagents are preferably provided without the presence of such attracting groups (though with the thermochemically reactive groups and optional photogroups). Suitable reagents of this type are disclosed in the above-captioned co-pending PCT application. Such reagents, in turn, can be used to coat oligonucleotides in a manner that provides an improved combination of such properties as reduced background, small spot size (e.g., increased contact angle), as compared to polymeric reagents having charged attracting groups.
The present invention provides a method and reagent composition for covalent attachment of target molecules onto the surface of a substrate, such as microwell plates, tubes, beads, microscope slides, silicon wafers or membranes. In one embodiment, the method and composition are used to immobilize nucleic acid probes onto plastic materials such as microwell plates, e.g., for use in hybridization assays. In a preferred embodiment the method and composition are adapted for use with substantially flat surfaces, such as those provided by microscope slides and other plastic, silicon hydride, or organosilane-pretreated glass or silicone slide support surfaces. The reagent composition can then be used to covalently attach a target molecule such as a biomolecule (e.g., a nucleic acid) which in turn can be used for specific binding reactions (e.g., to hybridize a nucleic acid to its complementary strand).
Support surfaces can be prepared from a variety of materials, including but not limited to plastic materials selected from the group consisting of crystalline thermoplastics (e.g., high and low density polyethylenes, polypropylenes, acetal resins, nylons and thermoplastic polyesters) and amorphous thermoplastics (e.g., polycarbonates and poly(methyl methacrylates). Suitable plastic or glass materials provide a desired combination of such properties as rigidity, toughness, resistance to long term deformation, recovery from deformation on release of stress, and resistance to thermal degradation.
A reagent composition of the invention contains one or more thermochemically reactive groups (i.e., groups having a reaction rate dependent on temperature). Suitable groups are selected from the group consisting of activated esters (e.g., NOS), epoxide, azlactone, activated hydroxyl and maleimide groups. Optionally, and preferably, the composition can also contain one or more photoreactive groups. Additionally, the reagent may comprise one or more hydrophilic polymers, to which the thermochemically reactive and/or photoreactive groups can be pendent. The photoreactive groups (alternatively referred to herein as xe2x80x9cphotogroupsxe2x80x9d) can be used, for instance, to attach reagent molecules to the surface of the support upon the application of a suitable energy source such as light. The thermochemically reactive groups, in turn, can be used to form covalent bonds with appropriate and complementary functional groups on the target molecule.
Generally, the reagent molecules will first be attached to the surface by activation of the photogroups, thereafter the target molecule, (e.g., an oligonucleotide) is contacted with the bound reagent under conditions suitable to permit it to come into binding proximity with the bound polymer. The target molecule is thermochemically coupled to the bound reagent by reaction between the reactive groups of the bound reagent and appropriate functional groups on the target molecule. The thermochemically reactive groups and the ionic groups can either be on the same polymer or, for instance, on different polymers that are coimmobilized onto the surface. Optionally, and preferably, the target molecule can be prepared or provided with functional groups tailored to groups of the reagent molecule. During their synthesis, for instance, the oligonucleotides can be prepared with functional groups such as amines or sulfhydryl groups.
The invention further provides a method of attaching a target molecule, such as an oligo, to a surface, by employing a reagent as described herein. In turn, the invention provides a surface having nucleic acids attached thereto by means of such a reagent, as well as a material (e.g., microwell plate) that provides such a surface. In yet another aspect, the invention provides a composition comprising a reagent(s) of this invention in combination with a target molecule that contains one or more functional groups reactive with the thermochemically reactive group(s) of the reagent.
Using such reagents, applicants have found that capture probes can be covalently immobilized to a variety of surfaces, including surfaces that would not otherwise adsorb the probes (such as polypropylene and polyvinylchloride). The resulting surfaces provide signals comparable to or better than those obtained with modified oligonucleotides adsorbed onto polystyrene or polycarbonate.
The present immobilization reagent and method can be used in amplification methods in a manner that is simpler than those previously reported, and can also provide improved surfaces for the covalent immobilization of nucleophile-derivatized nucleic acids. In addition to immobilized probes for amplification methods and hybridization assays, the reagents of this invention may provide improved nucleic acid immobilization for solid phase sequencing and for immobilizing primers for PCR and other amplification techniques.